The epidermal keratinocyte has received substantial attendance as a potential vehicle for introducing new genes into humans, and preliminary experimental results have been promising. There are many reasons for favorably considering keratinocytes, but technical and theoretical issues limit the usefulness of the keratinocyte gene therapy protocols reported to date. This pilot project tests the feasibility of inserting a new gene into keratinocytes by homologous recombination. If our preliminary results are successful, a full scale preclinical trial of the efficacy and practicality of this approach would be justified. Specific aims are: 1) Construct a targeting vector that will insert a positive selectable marker and the lacZ gene into the keratin 5 or the beta-actin locus; 2) transfect the targeting vector into normal human keratinocytes and determine the frequency of the vector's insertion and efficiency of homologous recombination has occurred and expand them into an in vitro epidermis in order to determine the duration and location of marker protein expression. This proposal represents a totally new line of investigation for Dr. Milstone. While gene targeting is Dr. Sedivy's main research interest, this proposal's focus on normal human keratinocytes involves methods for which a collaborative effort is necessary. As a pilot and feasibility study, we believe there is no overlap with Dr. Sedivy's currently funded project involving normal fibroblasts, since the feasibility largely depends on success with cell specific targeting vectors and cell specific optimization protocols.